Reagents and techniques that permit the specific detection of target molecules are important tools in many research areas and in clinical diagnostics. Many techniques have been developed for detecting target nucleic acid sequences, typically by detecting the hybridization of a complementary oligonucleotide probe sequence with a target sequence. These techniques often rely upon fluorescence resonance energy transfer (FRET), a phenomenon in which a change in fluorescence is caused by the interaction of two fluorophore groups.
One example of a class of oligonucleotide probes useful for detecting specific nucleic acid target sequences is referred to as “molecular beacons.” Molecular beacons are nucleic acid sequences that contain both a fluorophore and group that quenches the fluorescence of the fluorophore (Tyagi and Kramer, Nat. Biotech. 14:303-308, 1996). By design, molecular beacons yield “on/off” signals, and usage of molecular beacons is mainly in detecting DNA through hybridization. Molecular beacons are limited because the 3′ and 5′ donors and acceptors need to be within nanometer-scale proximity for effective energy transfer to occur.
In general the detection of molecules using molecular recognition events other than complementary oligonucleotide hybridization is a more difficult proposition. Indeed, no single method is universally applicable. Specific proteins can be detected by using antibody-based assays, such as an enzyme-linked immunosorbent assay (ELISA). However, this technique can only be used to detect molecules for which antibodies exist. Furthermore, it is time-consuming and expensive to generate new antibodies, particularly antibodies that bind to a specific target portion of the analyte molecule. If the antibody is not directed against the functional portion of the analyte molecule, for example, when the analyte is a protein, the ELISA may not be able to distinguish an active from an inactive protein. Moreover, many molecules of interest, such as small molecules, tend to be refractory to antibody production.